Adenomyosis (AM) is a common and difficult gynecological disease, but its specific mechanism has not been fully elucidated. Traditional Chinese Medicine (TCM) has certain advantages in the treatment of AM, preliminary studies have shown that Guilou compound-releasing intrauterine system can significantly reduce the proliferation and invasion ability of endometrial cells in rats with AM model.

To investigate the effect of Guilou compound on the migration and invasion of human adenomyosis derived cells (AMDC), and study its effect on Rho/ROCK signaling pathway.

The experiment was completed from October 2021 to April 2023 in Central Laboratory of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine. The CCK-8 assay was used to detect AMDC cell viability and screen the optimal drug concentration. AMDC were treated with Guilou compound (50 and 100 mg/L), and the cell migration viability was detected by scratch assay, and cell invasion ability was detected by Transwell assay. The expression of RhoA, RhoB, RhoC, ROCK1, and ROCK2 at protein and mRNA levels was detected by Western Blotting/RT-qPCR assay. In the Rescue experiment, AMDC were treated with agonist U-46619 (1 μmol/L) and Guilou compound (100 mg/L), and the protein expression of RhoA, RhoB, RhoC, ROCK1, and ROCK2 was detected by Western Blotting.

Compared with the control group, the survival rate of AMDC treated with 100 mg/L mass concentration of Guilou compound was not significantly decreased (P>0.05), which was the optimal drug concentration without affecting the cell viability, 50 and 100 mg/L mass concentrations of Guilou compound were selected for subsequent experiments. Compared with the control group, the migration and invasion ability of AMDC in the Guilou compound (50 mg/L, 100 mg/L) group was significantly inhibited (P<0.001), the protein and mRNA expression levels of RhoA, RhoC, ROCK1, and ROCK2 were significantly down-regulated (P<0.001), while the protein expression of RhoA, RhoC, ROCK1 and ROCK2 in the U-46619 1 μmol/L group were significantly up-regulated (P<0.001). Compared with the U-46619 1 μmol/L group, the protein expression of RhoA, RhoC, ROCK1, and ROCK2 in the U-46619 (1 μmol/L) combined with Guilou compound (100 mg/L) group was significantly down-regulated (P<0.001). There was no significant difference in protein and mRNA expression levels of RhoB in the control group and Guilou compound (50 mg/L, 100 mg/L) group (P>0.05) .

Guilou compound can effectively inhibit the migration and invasion ability of AMDC with the optimal drug concentration of 100 mg/L that does not affect the cell viability, Guilou compound can reverse the up-regulation of U-46619 on the protein expression of RhoA, RhoC, ROCK1 and ROCK2, thus inhibiting the the continuous progression of ectopic lesions of AM, which may be closely related to the regulation of Rho/ROCK pathway.

子宫腺肌病（AM）是妇科常见的疑难病，但其具体机制尚未明确。中医药治疗AM有一定优势，前期研究显示，桂楼复方宫内缓释系统可明显降低AM模型大鼠子宫内膜细胞增殖、侵袭能力。

探讨桂楼复方对子宫腺肌病异位内膜细胞（AMDC）迁移、侵袭的影响，并研究其对Rho/ROCK信号通路的影响。

本研究于2021年10月—2023年4月在山东中医药大学附属医院实验中心完成。使用CCK-8法检测AMDC细胞活力并筛选最佳药物浓度。设置空白组，使用桂楼复方（50、100 mg/L）处理AMDC，划痕实验检测细胞迁移能力，Transwell实验检测细胞侵袭能力，免疫印迹法Western Blotting）/实时荧光定量PCR（RT-qPCR）法检测RhoA、RhoB、RhoC、ROCK1、ROCK2在蛋白和mRNA水平的表达情况。Rescue实验中使用激动剂U-46619（1 μmol/L）和桂楼复方（100 mg/L）处理AMDC，Western Blotting法检测RhoA、RhoB、RhoC、ROCK1、ROCK2的蛋白表达改变情况。

与空白组比较，100 mg/L桂楼复方干预后AMDC细胞存活率未明显降低，差异无统计学意义（P>0.05），是不影响细胞活性的最佳药物浓度。选取50、100 mg/L桂楼复方进行后续实验。与空白组比较，桂楼复方50 mg/L、100 mg/L组AMDC迁移、侵袭能力受到抑制（P<0.001），RhoA、RhoC、ROCK1、ROCK2的蛋白及mRNA表达水平下调（P<0.001），U-46619 1 μmol/L组RhoA、RhoC、ROCK1、ROCK2的蛋白表达上调（P<0.001）。与U-46619 1 μmol/L组比较，U-46619 1 μmol/L联合桂楼复方100 mg/L组RhoA、RhoC、ROCK1、ROCK2的蛋白表达下调（P<0.001）。空白组与桂楼复方50 mg/L、100 mg/L组RhoB的蛋白及mRNA表达水平差异均无统计学意义（P>0.05）。

桂楼复方可有效抑制AMDC的迁移、侵袭能力，不影响细胞活性的最佳药物浓度为100 mg/L，桂楼复方可下调RhoA、RhoC、ROCK1、ROCK2的蛋白及mRNA表达水平，并可逆转U-46619对RhoA、RhoC、ROCK1、ROCK2蛋白表达水平的上调作用，从而抑制AM病局部异位病灶的持续进展，其作用可能与调控Rho/ROCK通路密切相关。